Polynucleotide constructs for increased lysine production

ABSTRACT

The invention relates to production of lysine, and provides several isolated polynucleotide molecules useful for the production of L-lysine. One such polynucleotide encodes an aspartate kinase (ask), an aspartate-semialdehyde dehydrogenase (asd) and a dihydrodipicolinate reductase (dapB). Other polypeptides encode ask, asd, dapB and a diaminopimelate dehydrogenase (ddh); ask, asd, dapB, ddh and an ORF2 poypeptide; and ask, asd dapB, ddh, ORF2 and a diaminopimelate decarboxylase (lysA). The invention further provides methods of making and using the polynucleotides, and methods to increase the production of L-lysine.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the right of priority under 35 U.S.C. §119 toU.S. Provisional Appl. No. 60/267,183 filed on Feb. 8, 2001, theentirety of which is incorporated by reference herein.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The invention relates to production of lysine, and provides severalisolated polynucleotide molecules useful for the production of L-lysine.One such polynucleotide encodes an aspartate kinase (ask), anaspartate-semialdehyde dehydrogenase (asd) and a dihydrodipicolinatereductase (dapB). Other polypeptides encode ask, asd, dapB and adiaminopimelate dehydrogenase (ddh); ask, asd, dapB, ddh and an ORF2polypeptide; and ask, asd dapB, ddh, ORF2 and a diaminopimelatedecarboxylase (lysA). The invention further provides methods of makingand using the polynucleotides, and methods to increase the production ofL-lysine.

2. Related Art

L-lysine is an important economic product obtained principally byindustrial-scale fermentation utilizing the Gram positiveCorynebacterium glutamicum, Brevibacterium flavum and Brevibacteriumlactofermentum (Kleemann, A., el. al., Amino Acids, in ULLMANN'SENCYCLOPEDIA OF INDUSTRIAL CHEMISTRY, vol. A2, pp. 57-97, Weinham:VCH-Verlagsgesellschaft (1985)).

The stereospecificity of the amino acids produced by fermentation makesthe process advantageous compared with synthetic processes; generallyL-form amino acids are produced by the microbial fermentation process.The production of L-lysine and other amino acids through fermentation,utilizing cheap carbon sources such as molasses, glucose, acetic acidand ethanol, is a relatively inexpensive means of production.

Several fermentation processes utilizing various strains isolated forauxotrophic or resistance properties are known in the art for theproduction of L-lysine: U.S. Pat. No. 2,979,439 discloses mutantsrequiring amino acid supplementation (homoserine, or L-methionine andL-threonine); U.S. Pat. No. 3,700,557 discloses mutants having anutritional requirement for L-threonine, L-methionine, L-arginine,L-histidine, L-leucine, L-isoleucine, L-phenylalanine, L-cystine, orL-cysteine; U.S. Pat. No. 3,707,441 discloses a mutant having aresistance to an L-lysine analog; U.S. Pat. No. 3,687,810 discloses amutant having both an ability to produce L-lysine and a resistance tobacitracin, penicillin G or polymyxin; U.S. Pat. No. 3,708,395 disclosesmutants having a nutritional requirement for homoserine, L-threonine,L-threonine and L-methionine, L-leucine, L-isoleucine or mixturesthereof and a resistance to L-lysine, L-threonine, L-isoleucine oranalogs thereof, U.S. Pat. No. 3,825,472 discloses a mutant having aresistance to an L-lysine analog; U.S. Pat. No. 4,169,763 disclosesmutant strains of Corynebacterium that produce L-lysine and areresistant to at least one of aspartic analogs and sulfa drugs; U.S. Pat.No. 5,846,790 discloses a mutant strain able to produce L-glutamic acidand L-lysine in the absence of any biotin action-suppressing agent; andU.S. Pat. No. 5,650,304 discloses a strain belonging to the genusCorynebacterium or Brevibacterium for the production of L-lysine that isresistant to 4-N-(D-alanyl)-2,4-diamino-2,4-dideoxy-L-arabinose2,4-dideoxy-L-arabinose or a derivative thereof.

A considerable amount is known regarding the biochemical pathway forL-lysine synthesis in Corynebacterium species (recently reviewed by Sahmet al., Ann. N. Y. Acad. Sci. 782: 25-39 (1996)). Entry into theL-lysine pathway begins with L-aspartate (see FIG. 1), which itself isproduced by transamination of oxaloacetate. A special feature of C.glutamicum is its ability to convert the L-lysine intermediatepiperidine 2,6-dicarboxylate to diaminopimelate by two different routes,i.e. by reactions involving succinylated intermediates or by the singlereaction of diaminopimelate dehydrogenase. Overall, carbon flux into thepathway is regulated at two points: first, through feedback inhibitionof aspartate kinase by the levels of both L-threonine and L-lysine; andsecond through the control of the level of dihydrodipicolinate synthase.Therefore, increased production of L-lysine can be obtained inCorynebacterium species by deregulating and increasing the activity ofthese two enzymes.

More recent developments in the area of L-lysine fermentative productioninvolve the use of molecular biology techniques to augment L-lysineproduction. The following examples are provided: U.S. Pat. Nos.4,560,654 and 5,236,831 disclose an L-lysine producing mutant strainobtained by transforming a host Corynebacterium or Brevibacteriumspecies microorganism which is sensitive to S-(2-aminoethyl)-cysteinewith a recombinant DNA molecule wherein a DNA fragment conferring bothresistance to S-(2-aminoethyl)-cysteine and L-lysine producing abilityis inserted into a vector DNA; U.S. Pat. No. 5,766,925 discloses amutant strain produced by integrating a gene coding for aspartokinase,originating from coryneform bacteria, with desensitized feedbackinhibition by L-lysine and L-threonine, into chromosomal DNA of aCorynebacterium species bacterium harboring leaky type homoserinedehydrogenase or a Corynebacterium species deficient in homoserinedehydrogenase gene; increased L-lysine production is obtained by geneamplification by way of a plasmid vector or utilizing a gene replacementstrategy. European Patent Applications EP 0 811 682 A2 and EP 0 854 189A2 both provide for increased production of L-lysine in Corynebacteriumspecies by way of gene amplification based on plasmid copy number.

SUMMARY OF THE INVENTION

It is an object of the invention to provide an isolated polynucleotidemolecule, referred herein as the KDB polynucleotide, comprising anucleic acid molecule encoding an aspartate kinase (ask) polypeptide; anucleic acid molecule encoding an aspartate-semialdehyde dehydrogenase(asd) polypeptide and a nucleic acid molecule encoding adihydrodipicolinate reductase (dapB) polypeptide. The polynucleotide mayfurther comprise a nucleic acid encoding a complete or truncateddiaminopimelate dehydrogenase (ddh) polypeptide (the KDBHpolynucleotide), or a nucleic acid encoding a complete or truncated ORF2polypeptide (the KDB2 polynucleotide). In addition, the inventionprovides an isolated polynucleotide molecule, referred herein as theKDB2HL polynucleotide, comprising a nucleic acid molecule encoding anask, asd, dapB, ddh, ORF2 and diaminopimelate decarboxylase (lysA)polypeptides, in which the ddh, ORF2 and lysA polypeptides may becomplete or truncated. In a preferred embodiment, a polynucleotidemolecule of the invention further comprises a P1 promoter adjacent tothe 5′ end of the nucleotide molecule encoding lysA.

It is further the object of the invention to provide a method ofincreased L-lysine production in a host cell by transforming a host cellwith the polynucleotide molecules described above. According to themethod of the present invention, the isolated polynucleotide moleculesdescribed above are stably integrated into a chromosome of the hostcell, or are maintained as extrachromosomal DNA, such as a plasmid, andthe transformed host cells are selected for increased L-lysineproduction.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. A schematic of the L-lysine biosynthetic pathway inCorynebacterium glutamicum.

FIG. 2 A, B. The nucleotide (SEQ ID NO:1) and amino acid sequence (SEQID NO:2) of ask (ATCC 21529 sequence).

FIG. 3 A, B. The nucleotide (SEQ ID NO:3) and amino acid sequence (SEQID NO:4) of asd (ATCC 21529 sequence).

FIG. 4. The nucleotide (SEQ ID NO:5) and amino acid sequence (SEQ ID.NO:6) of dapB (NRRL-B11474).

FIG. 5 A, B. The nucleotide (SEQ ID NO:7) and amino acid sequence (SEQID NO:8) of ddh (NRRL-B11474).

FIG. 6. The nucleotide (SEQ ID NO:9) and amino acid sequence (SEQ ID NO:10) of ORF2.

FIG. 7 A, B, C. The nucleotide (SEQ ID NO: 11) and amino acid sequence(SEQ ID NO: 12) of lysA.

FIG. 8. The nucleotide (SEQ ID NO: 13) and amino acid sequence (SEQ IDNO: 14) of truncated ORF2.

FIG. 9. The nucleotide sequence (SEQ ID NO: 15) of the P1 promoter, thefirst promoter of the argS-lysA operon from pRS6.

FIG. 10. Comparison of the aspartokinase (ask) amino acid sequence fromATCC13032, N13 and ATCC21529.

FIGS. 11A and B. A schematic of the construction of thepDElia2_(FC5)-KDB construct.

FIG. 12. A schematic of the construction of the pK184-KDBH construct.

FIG. 13. A schematic of the construction of the pDElia2_(FC5)-KDB2construct.

FIG. 14 A, B. A schematic of the construction of thepDElia2_(FC5)-KDB2HP1 L construct.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

A. Definitions

In order to provide a clear and consistent understanding of thespecification and claims, including the scope to be given such terms,the following definitions are provided. It is also to be noted that theterm “a” or “an” entity, refers to one or more of that entity; forexample, “a polynucleotide,” is understood to represent one or morepolynucleotides.

Auxotroph. As used herein, the term refers to a strain of microorganismrequiring for growth an external source of a specific metabolite thatcannot be synthesized because of an acquired genetic defect.

Amino Acid Supplement. As used herein, the term refers to an amino acidrequired for growth and added to minimal media to support auxotrophgrowth.

Chromosomal Integration. As used herein, the term refers to theinsertion of an exogenous DNA fragment into the chromosome of a hostorganism; more particularly, the term is used to refer to homologousrecombination between an exogenous DNA fragment and the appropriateregion of the host cell chromosome.

High Yield Derivative. As used herein, the term refers to strain ofmicroorganism that produces a higher yield from dextrose of a specificamino acid when compared with the parental strain from which it isderived.

Host Cell. As used herein, the term “host cell” is intended to beinterchangeable with the term “microorganism.” Where a difference isintended, the difference will be made clear.

Isolated Nucleic Acid Molecule. As used herein, the term is intended tomean a nucleic acid molecule, DNA or RNA, which has been removed fromits native environment. For example, recombinant DNA molecules containedin a vector are considered isolated for the purposes of the presentinvention. Further examples of isolated DNA molecules includerecombinant DNA molecules maintained in heterologous host cells orpurified (partially or substantially) DNA molecules in solution.Isolated RNA molecules include in vivo or in vitro RNA transcripts ofthe DNA molecules of the present invention. Isolated nucleic acidmolecules according to the present invention further include suchmolecules produced synthetically.

Lysine Biosynthetic Pathway Genes. As used herein, the term “lysinebiosynthetic pathway gene(s)” is meant to include those genes and genesfragments encoding peptides, polypeptides, proteins, and enzymes, whichare directly involved in the synthesis of lysine. These genes can beidentical to those which naturally occur within a host cell and areinvolved in the synthesis of lysine within that host cell.Alternatively, there can be modifications or mutations of such genes,for example, the genes can contain modifications or mutations which donot significantly affect the biological activity of the encoded protein.For example, the natural gene can be modified by mutagenesis or byintroducing or substituting one or more nucleotides or by removingnonessential regions of the gene. Such modifications are readilyperformed by standard techniques.

Lysine Biosynthetic Pathway Protein. As used herein, the term “lysinebiosynthetic pathway protein” is meant to include those peptides,polypeptides, proteins, and enzymes, which are directly involved in thesynthesis of lysine from aspartate. Also included are amino acidsequences as encoded by open reading frames (ORF), where the ORF isassociated with a lysine biosynthetic pathway operon. These proteins canbe identical to those which naturally occur within a host cell and areinvolved in the synthesis of lysine within that host cell.Alternatively, there can be modifications or mutations of such proteins,for example, the proteins can contain modifications or mutations whichdo not significantly affect the biological activity of the protein. Forexample, the natural protein can be modified by mutagenesis or byintroducing or substituting one or more amino acids, preferably byconservative amino acid substitution, or by removing nonessentialregions of the protein. Such modifications are readily performed bystandard techniques. Alternatively, lysine biosynthetic proteins can beheterologous to the particular host cell. Such proteins can be from anyorganism having genes encoding proteins having the same, or similar,biosynthetic roles.

Mutagenesis. As used herein, the term refers to a process whereby amutation is generated in DNA. With “random” mutagenesis, the exact siteof mutation is not predictable, occurring anywhere in the genome of themicroorganism, and the mutation is brought about as a result of physicaldamage caused by agents such as radiation or chemical treatment. rDNAmutagenesis is directed to a cloned DNA of interest, and it can berandom or site-directed.

Mutation. As used herein, the term refers to a one or more base pairchange, insertion or deletion, or a combination thereof, in thenucleotide sequence of interest.

Operably Linked. As used herein, the term “operably linked” refers to alinkage of polynucleotide elements in a functional relationship. Anucleic acid is “operably linked” when it is placed into a functionalrelationship with another nucleic acid sequence. For instance, apromoter or enhancer is operably linked to a coding sequence if itaffects the transcription of the coding sequence. Operably linked meansthat the DNA sequences being linked are typically contiguous and, wherenecessary, join two protein coding regions, contiguous and in readingframe. However, since enhancers generally function when separated fromthe promoter by several kilobases and intronic sequences can be ofvariable lengths, some polynucleotide elements can be operably linkedbut not contiguous.

Operon. As used herein, the term refers to a contiguous portion of atranscriptional complex in which two or more open reading framesencoding polypeptides are transcribed as a multi-cistronic messengerRNA, controlled by a cis-acting promoter and other cis-acting sequencesnecessary for efficient transcription, as well as additional cis actingsequences important for efficient transcription and translation (e.g.,mRNA stability controlling regions and transcription terminationregions). The term generally also refers to a unit of gene expressionand regulation, including the structural genes and regulatory elementsin DNA.

Parental Strain. As used herein, the term refers to a strain of hostcell subjected to some form of treatment to yield the host cell of theinvention.

Percent Yield From Dextrose. As used herein, the term refers to theyield of amino acid from dextrose defined by the formula [(g amino acidproduced/g dextrose consumed)*100]=% Yield.

Phenotype. As used herein, the term refers to observable physicalcharacteristics dependent upon the genetic constitution of a host cell.

Promoter. As used herein, the term “promoter” has its art-recognizedmeaning, denoting a portion of a gene containing DNA sequences thatprovide for the binding of RNA polymerase and initiation oftranscription and thus refers to a DNA sequence capable of controllingthe expression of a coding sequence or functional RNA. Promotersequences are commonly, but not always, found in the 5′ non-codingregions of genes. In general, a coding sequence is located 3′ to apromoter sequence. Sequence elements within promoters that function inthe initiation of transcription are often characterized by consensusnucleotide sequences. The promoter sequence consists of proximal andmore distal upstream elements (enhancers). As used herein, the term“endogenous promoter” refers to a promoter sequence which is a naturallyoccurring promoter sequence in that host microorganism. The term“heterologous promoter” refers to a promoter sequence which is anon-naturally occurring promoter sequence in that host microorganism.The heterologous occurring promoter sequence can be from any prokaryoticor eukaryotic organism. A synthetic promoter is a nucleotide sequence,having promoter activity, and not found naturally occurring in nature.

Promoters can be derived in their entirety from a native gene, or behybrid promoters. Hybrid promoters are composed of different elementsderived from different promoters found in nature, or even comprisesynthetic DNA segments. Hybrid promoters can be constitutive, inducibleor environmentally responsive.

Useful promoters include constitutive and inducible promoters. Many suchpromoter sequences are known in the art. See, for example, U.S. Pat.Nos. 4,980,285; 5,631,150; 5,707,828; 5,759,828; 5,888,783; 5,919,670,and, Sambrook, et al., Molecular Cloning: A Laboratory Manual, 2nd Ed.,Cold Spring Harbor Press (1989). Other useful promoters includepromoters which are neither constitutive nor responsive to a specific(or known) inducer molecule. Such promoters can include those thatrespond to developmental cues (such as growth phase of the culture), orenvironmental cues (such as pH, osmoticum, heat, or cell density, forexample).

Examples of environmental conditions that can affect transcription byinducible promoters include anaerobic conditions, elevated temperature,or the presence of light. It is understood by those skilled in the artthat different promoters can direct the expression of a gene indifferent cell types, or in response to different environmentalconditions. Promoters which cause a gene to be expressed in most celltypes at most times are commonly referred to as “constitutivepromoters.” It is further recognized that since in most cases the exactboundaries of regulatory sequences have not been completely defined, DNAfragments of different lengths can have identical or similar promoteractivity.

Relative Growth. As used herein, the term refers to a measurementproviding an assessment of growth by directly comparing growth of aparental strain with that of a progeny strain over a defined time periodand with a defined medium.

B. Microbiological and Recombinant DNA Methodologies

The present invention relates to a KDB polynucleotide, which comprises anucleic acid encoding an ask polypeptide, a nucleic acid moleculeencoding an asd polypeptide and a nucleic acid molecule encoding a dapBpolypeptide, wherein “K” represents a nucleotide sequence encoding theask polypeptide; “D” represents a nucleotide sequence encoding the asdpolypeptide; and “B” represents a nucleotide sequence encoding the dapBpolypeptide.

In one embodiment the present invention relates to an isolated KDBpolynucleotide molecule comprising:

-   -   1. a nucleic acid molecule encoding an aspartate kinase (ask)        polypeptide;    -   2. a nucleic acid molecule encoding an aspartate-semialdehyde        dehydrogenase (asd) polypeptide; and    -   3. a nucleic acid molecule encoding a dihydrodipicolinate        reductase (dapB) polypeptide.

In another embodiment, the KDB polynucleotide molecule consistsessentially of a nucleic acid molecule encoding an ask polypeptide, anucleic acid molecule encoding an asd polypeptide and a nucleic acidmolecule encoding a dapB polypeptide.

The present invention also relates to a KDBH polynucleotide, whichcomprises a nucleic acid encoding an ask polypeptide, a nucleic acidmolecule encoding an asd polypeptide, a nucleic acid molecule encoding adapB polypeptide and a nucleic acid molecule encoding a ddh polypeptide,wherein “K” represents a nucleotide sequence encoding the askpolypeptide; “D” represents a nucleotide sequence encoding the asdpolypeptide; “B” represents a nucleotide sequence encoding the dapBpolypeptide; and “H” represents a nucleotide sequence encoding the ddhpolypeptide.

In one embodiment, the KDBH polynucleotide molecule additionallycomprises a nucleic acid encoding a complete or truncated ORF2polypeptide.

The present invention also relates to a KDB2 polynucleotide, whichcomprises a nucleic acid encoding an ask polypeptide, a nucleic acidmolecule encoding an asd polypeptide, a nucleic acid molecule encoding adapB polypeptide and a nucleic acid molecule encoding an ORF2polypeptide, and wherein “K” represents a nucleotide sequence encodingthe ask polypeptide; “D” represents a nucleotide sequence encoding theasd polypeptide; “B” represents a nucleotide sequence encoding the dapBpolypeptide; and “2” represents a nucleotide sequence encoding the ORF2polypeptide.

The present invention also relates to a KDB2HL polynucleotide, whichcomprises a nucleotide encoding an ask polypeptide, a nucleic acidmolecule encoding an asd polypeptide, a nucleic acid molecule encoding adapB polypeptide, a nucleic acid molecule encoding an ORF2 polypeptide,a nucleic acid molecule encoding a ddh polypeptide and a nucleic acidmolecule encoding a lysA polypeptide. In a preferred embodiment, theKDB2HL polynucleotide molecule also comprises a P1 promoter adjacent tothe 5′ end of the nucleic acid encoding the lysA polypeptide.

In a preferred embodiment, the polynucleotide molecules of the presentinvention do not comprise any nucleic acid molecules encoding any lysinepathway polypeptides other than ask, asd, dapB, ddh, ORF2 and lysA.

In one embodiment, an ask polypeptide is defined as a polypeptide havingthe enzymatic activity of bacterial aspartate kinase. Bacterialaspartate kinase enzymatic activity converts L-aspartate toL-aspartylphosphate. In a preferred embodiment, an ask polypeptide wouldhave the enzymatic activity of aspartate kinase from ATCC21529. In apreferred embodiment, the isolated ask amino sequence disclosed in SEQID NO:2 possesses unique properties with respect to feedback resistanceof ask enzyme activity to accumulated levels of L-lysine and L-threoninein the culture medium. When compared to the DNA sequences of otherCorynebacterium glutamicum ask-asd gene sequences, a threonine toisoleucine change at amino acid residue 380 which results in resistanceto feedback inhibition is observed. Other amino acid changes at residue380 can also result in decreased ask enzyme sensitivity to L-threonineand/or L-lysine.

An asd polypeptide is defined as a polypeptide having the enzymaticactivity of aspartate-semialdehyde dehydrogenase. Aspartate-semialdehydedehydrogenase enzymatic activity converts L-aspartylphosphate toL-aspartatesemialdehyde. In a preferred embodiment, an asd polypeptidewould have the enzymatic activity of aspartate-semialdehydedehydrogenase from ATCC21529.

A dapB polypeptide is defined as a polypeptide having the enzymaticactivity of dihydrodipicolinate reductase. Dihydrodipicolinate reductaseenzymatic activity converts L-2,3-dihydrodipicolinate toL-piperideine-2,6-dicarboxylate. In a preferred embodiment, a dapBpolypeptide would have the enzymatic activity of dihydrodipicolinatereductase from NRRL-B11474.

A ddh polypeptide is defined as a polypeptide having the enzymaticactivity of diaminopimelate dehydrogenase. Diaminopimelate dehydrogenaseenzymatic activity converts L-piperideine-2,6-dicarboxylate toD,L-diaminopimelate. In a preferred embodiment, a ddh polypeptide wouldhave the enzymatic activity of diaminopimelate dehydrogenase fromNRRL-B11474.

A lysA polypeptide is defined as a polypeptide having the enzymaticactivity of diaminopimelate decarboxylase. Diaminopimelate decarboxylaseactivity converts D,L-diaminopimelate to L-lysine. In a preferredembodiment, a lysA polypeptide would have the enzymatic activity ofdiaminopimelate decarboxylase from ASO19.

Ask, asd, dapB, ddh, ORF2 and lysA polypeptides encoded by thepolynucleotide molecules of the present invention can be truncated formsof the polypeptides encoded by the genomic copies of the ATCC21529 askand asd genes, the NRRL-B11474 dapB, ddh, ORF2 genes and the ASO19 lysAgene.

It should be noted that in addition to the indicated polypeptidesequences encoded by the isolated nucleic acid sequences represented by“K”, “D”, “B”, “H,” “2” and “L,” these isolated nucleic acid sequencescan also include native promoter elements for the operons representedtherein. Thus, the ask-asd sequences can include the respective nativeask-asd operon elements, and the dapB and ddh sequences can includetheir respective native promoter elements. The preferred promoter forthe nucleotide molecule encoding the lysA polypeptide is the P1promoter, the first promoter from the argS-lysA operon.

The invention as provided herein utilizes some methods and techniquesthat are known to those skilled in the arts of microbiology andrecombinant DNA technologies. Methods and techniques for the growth ofbacterial cells, the introduction of isolated DNA molecules into hostcells, and the isolation, cloning and sequencing of isolated nucleicacid molecules, etc., are a few examples of such methods and techniques.These methods and techniques are described in many standard laboratorymanuals, such as Davis et al., Basic Methods In Molecular Biology(1986), J. H. Miller, Experiments in Molecular Genetics, Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y. (1972); J. H. Miller,A Short Course in Bacterial Genetics, Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y. (1992); M. Singer and P. Berg, Genes &Genomes, University Science Books, Mill Valley, Calif. (1991); J.Sambrook, E. F. Fritsch and T. Maniatis, Molecular Cloning: A LaboratoryManual, 2d ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor,N.Y. (1989); P. B. Kaufman et al., Handbook of Molecular and CellularMethods in Biology and Medicine, CRC Press, Boca Raton, Fla. (1995);Methods in Plant Molecular Biology and Biotechnology, B. R. Glick and J.E. Thompson, eds., CRC Press, Boca Raton, Fla. (1993); and P. F.Smith-Keary, Molecular Genetics of Escherichia coli, The Guilford Press,New York, N.Y. (1989), all of which are incorporated herein by referencein their entireties.

In a preferred embodiment, a nucleic acid molecule encoding an askpolypeptide would be at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98% or 99% identical to SEQ ID NO:1. A nucleic acid molecule encoding anasd polypeptide would be at least 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98% or 99% identical to SEQ ID NO:3. a nucleic acid moleculeencoding a dapB polypeptide would be at least 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:5, a nucleic acidmolecule encoding a ddh polypeptide would be at least 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:7, a nucleicacid encoding an ORF2 polypeptide would be at least 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:9 and anucleic acid encoding a lysA polypeptide would be at least 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:11or the complement thereof. In a preferred embodiment the nucleic acidsequence of a P1 promoter would be at least 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:15. In oneembodiment, a nucleic acid encoding a truncated ORF2 polypeptide wouldbe at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%identical to SEQ ID NO:13.

As one skilled in the art would know, any strain of Corynebacteriumspecies, particularly that of Corynebacterium glutamicum, can beutilized for the isolation of nucleic acid molecules that will be usedto amplify the number of chromosomally located amino acid biosyntheticpathway genes. Particularly preferred strains include: NRRL-B11474, ATCC21799, ATCC 21529, ATCC 21543, and E12.

As a practical matter, whether any particular nucleic acid sequence isat least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identicalto, for instance, a nucleotide sequence consisting of SEQ ID NO:1, SEQID NO:3; SEQ ID NO:5; SEQ ID NO:7; SEQ ID NO:9; SEQ ID NO:11; SEQ IDNO:13; SEQ ID NO:15, or a complementary sequence thereof, can bedetermined conventionally using sequence analysis computer programs suchas a OMIGA® Version 2.0 for Windows, available from Oxford Molecular,Ltd. (Oxford, U.K.). OMIGA uses the CLUSTAL W alignment algorithm usingthe slow full dynamic programming alignment method with defaultparameters of an open gap penalty of 10 and an extend gap penalty of5.0, to find the best alignment between two nucleotide sequences. Whenusing CLUSTAL W or any other sequence alignment program to determinewhether a particular sequence is, for instance, 95% identical to areference sequence according to the present invention, the parametersare set, of course, such that the percentage of identity is calculatedover the full length of the reference nucleotide sequence such thatgaps, mismatches, or insertions of up to 5% of the total number ofnucleotides in the reference sequence are allowed. Other sequenceanalysis programs, known in the art, can be used in the practice of theinvention.

Unless otherwise indicated, all nucleotide sequences described hereinwere determined using an automated DNA sequencer (such as the Model 373from Applied Biosystems, Inc.), and all amino acid sequences ofpolypeptides encoded by DNA molecules described herein were predicted bytranslation of the relative DNA sequence. Therefore, as is known in theart, for any DNA sequence determined by this automated approach, anynucleotide sequence determined herein can contain some errors.Nucleotide sequences determined by automation are typically at leastabout 90% identical, more typically at least about 95% to at least about99.9% identical to the actual nucleotide sequence of the sequenced DNAmolecule. The actual sequence can be more precisely determined by otherapproaches including manual DNA sequencing methods well known in theart.

It is known in the art that amino acids are encoded at the nucleic acidlevel by one or more codons (code degeneracy). It is also known in theart that choice of codons may influence expression of a particular aminoacid sequence (protein, polypeptide, etc.). Thus, the invention isfurther directed to nucleic acid molecules encoding the ask amino acidsequence of SEQ ID NO:2 wherein the nucleic acid molecule comprises anycodon known to encode a particular amino acid. Likewise, the inventionis directed to KDB, KDBH, KDB2 and KDB2HL polynucleotides comprisingnucleic acid sequences which comprise alternative codons in order tooptimize expression of the protein or polypeptide.

It will be recognized in the art that some amino acid sequences of theinvention can be varied without significant effect of the structure orfunction of the proteins disclosed herein. Variants included canconstitute deletions, insertions, inversions, repeats, and typesubstitutions so long as enzyme activity is not significantly affected.Guidance concerning which amino acid changes are likely to bephenotypically silent can be found in Bowie, J. U., et al., “Decipheringthe Message in Protein Sequences: Tolerance to Amino AcidSubstitutions,” Science 247:1306-1310 (1990).

It is preferred that the polypeptides obtained by the expression of thepolynucleotide molecules of the present invention would have at leastapproximately 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity to oneor more amino acid sequences selected from the group comprising SEQ IDNo: 2, 4, 6, 8, 10, 12 and 14. A truncated ORF2 polypeptide has at leastabout 25% of the full length of an ORF2 polypeptide, preferably the ORF2polypeptide of SEQ ID NO: 10. In one embodiment, a truncated ORF2polypeptide has the sequence of SEQ ID NO: 14. By a polypeptide havingan amino acid sequence at least, for example, 95% “identical” to areference amino acid sequence of a polypeptide is intended that theamino acid sequence of the claimed polypeptide is identical to thereference sequence except that the claimed polypeptide sequence caninclude up to five amino acid alterations per each 100 amino acids ofthe reference amino acid of the polypeptide. In other words, to obtain apolypeptide having an amino acid sequence at least 95% identical to areference amino acid sequence, up to 5% of the amino acid residues inthe reference sequence can be deleted or substituted with another aminoacid, or a number of amino acids up to 5% of the total amino acidresidues in the reference sequence can be inserted into the referencesequence. These alterations of the reference sequence can occur at theamino or carboxy terminal positions of the reference amino acid sequenceor anywhere between those terminal positions, interspersed eitherindividually among residues in the reference sequence or in one or morecontiguous groups within the reference sequence.

As a practical matter, whether any particular polypeptide is at least80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to, forinstance, the amino acid sequence shown in SEQ ID NO:2, 4, 6, 8, 10, 12,14 or to the amino acid sequence encoded by a nucleic acid sequence canbe determined conventionally using known computer programs such theBestfit program (Wisconsin Sequence Analysis Package, Version 8 forUnix, Genetics Computer Group, University Research Park, 575 ScienceDrive, Madison, Wis. 53711). When using Bestfit or any other sequencealignment program to determine whether a particular sequence is, forinstance, 95% identical to a reference sequence according to the presentinvention, the parameters are set, of course, such that the percentageof identity is calculated over the full length of the reference aminoacid sequence and that gaps in homology of up to 5% of the total numberof amino acid residues in the reference sequence are allowed.

In a specific embodiment, the identity between a reference sequence(query sequence, a sequence of the present invention) and a subjectsequence, also referred to as a global sequence alignment, is determinedusing the FASTDB computer program based on the algorithm of Brutlag etal. (Comp. App. Biosci. 6:237-245 (1990)). Preferred parameters used ina FASTDB amino acid alignment are: Matrix=PAM 0, k-tuple=2, MismatchPenalty=1, Joining Penalty=20, Randomization Group Length=0, CutoffScore=1, Window Size=sequence length, Gap Penalty=5, Gap SizePenalty=0.05, Window Size=500 or the length of the subject amino acidsequence, whichever is shorter. According to this embodiment, if thesubject sequence is shorter than the query sequence due to N- orC-terminal deletions, not because of internal deletions, a manualcorrection is made to the results to take into consideration the factthat the FASTDB program does not account for N- and C-terminaltruncations of the subject sequence when calculating global percentidentity. For subject sequences truncated at the N- and C-termini,relative to the query sequence, the percent identity is corrected bycalculating the number of residues of the query sequence that are N- andC-terminal of the subject sequence, which are not matched/aligned with acorresponding subject residue, as a percent of the total bases of thequery sequence. A determination of whether a residue is matched/alignedis determined by results of the FASTDB sequence alignment. Thispercentage is then subtracted from the percent identity, calculated bythe above FASTDB program using the specified parameters, to arrive at afinal percent identity score. This final percent identity score is whatis used for the purposes of this embodiment. Only residues to the N- andC-termini of the subject sequence, which are not matched/aligned withthe query sequence, are considered for the purposes of manuallyadjusting the percent identity score. That is, only query residuepositions outside the farthest N- and C-terminal residues of the subjectsequence. For example, a 90 amino acid residue subject sequence isaligned with a 100 residue query sequence to determine percent identity.The deletion occurs at the N-terminus of the subject sequence andtherefore, the FASTDB alignment does not show a matching/alignment ofthe first 10 residues at the N-terminus. The 10 unpaired residuesrepresent 10% of the sequence (number of residues at the N- andC-termini not matched/total number of residues in the query sequence) so10% is subtracted from the percent identity score calculated by theFASTDB program. If the remaining 90 residues were perfectly matched thefinal percent identity would be 90%. In another example, a 90 residuesubject sequence is compared with a 100 residue query sequence. Thistime the deletions are internal deletions so there are no residues atthe N- or C-termini of the subject sequence which are notmatched/aligned with the query. In this case the percent identitycalculated by FASTDB is not manually corrected. Once again, only residuepositions outside the N- and C-terminal ends of the subject sequence, asdisplayed in the FASTDB alignment, which are not matched/aligned withthe query sequence are manually corrected for.

C. Methods and Processes of the Invention

Various embodiments of the invention provide methods of utilizing theKDB, KDBH, KDB2 and KDB2HL polynucleotide molecules. In a preferredembodiment, any one of these polynucleotide molecules is utilized toincrease the production of lysine from a host cell.

The amino acid pathway for L-lysine biosynthesis is well known toskilled artisans of amino acid production. Genes encoding the enzymesimportant for the conversion of L-aspartate to L-lysine include the ask,asd, dapA, dapB, ddh and lysA genes (FIG. 1). Thus, the inventionprovides herein specific embodiments utilizing L-lysine biosyntheticpathway genes.

The isolated polynucleotide molecules of the invention are preferablypropagated and maintained in an appropriate nucleic acid vector. Methodsfor the isolation and cloning of the isolated nucleic acid molecules ofthe invention are well known to those skilled in the art of recombinantDNA technology. Appropriate vectors and methods for use with prokaryoticand eukaryotic hosts are described by Sambrook et al., MolecularCloning: A Laboratory Manual, Second Edition, Cold Spring Harbor, N.Y.,1989, the disclosure of which is hereby incorporated by reference.

A great variety of vectors can be used in the invention. Such vectorsinclude chromosomal, episomal and virus-derived vectors, e.g., vectorsderived from bacterial plasmids and from bacteriophage, as well asvectors derived from combinations thereof, such as those derived fromplasmid and bacteriophage genetic elements, such as cosmids andphagemids, all can be used in accordance with this aspect of the presentinvention. Retroviral vectors can be replication competent orreplication defective. In the latter case, viral propagation generallywill occur only in complementing host cells. Preferred, are vectorssuitable to maintain and propagate a polynucleotide in a bacterial host.

A large numbers of suitable vectors and promoters for use in bacteriaare known, many of which are commercially available. Preferredprokaryotic vectors include plasmids such as those capable ofreplication in E. coli (such as, for example, pBR322, ColE1, pSC101,pACYC 184, πVX). Such plasmids are, for example, disclosed by Maniatis,T., et al., In. Molecular Cloning, A Laboratory Manual, Cold SpringHarbor Press, Cold Spring Harbor, N.Y. (1982)). The following vectorsare provided by way of example: pET (Novagen), pQE70, pQE60, pQE-9(Qiagen), pBs, phagescript, psiX174, pBlueScript SK, pBsKS, pNH8a,pNH16a, pNH18a, pNH46a (Stratagene), pTrc99A, pKK223-3, pKK233-3,pDR540, pRIT5 (Pharmacia).

Preferred vectors for the isolated nucleic acid molecules of theinvention include the pFC1 to pFC7 novel family of combinatorial cloningvectors (Lonsdale, D. M., et al., Plant Molecular Biology Reporter 13:343-345 (1995)) and the pK184 vector (Jobling, M. G. and Homes, R. K.,Nucleic Acid Research 18: 5315-5316 (1990)).

Another group of preferred vectors are those that are capable ofautonomous replication in Corynebacterium species. Such vectors are wellknown to those skilled in the art of amino acid production by way ofmicrobial fermentation, examples of which include pSR1, pMF1014α andvectors derived therefrom. Other suitable vectors will be readilyapparent to the skilled artisan.

A KDB, KDBH, KDB2 or KDB2HL polynucleotide can be joined to a vectorcontaining a selectable marker for propagation in a host. The vectorscan include at least one selectable marker. In this regard, vectorspreferably contain one or more selectable marker genes to provide aphenotypic trait for selection of transformed host cells. Such markersinclude dihydrofolate reductase, G418 or neomycin resistance foreukaryotic cell culture and tetracycline, kanamycin or ampicillinresistance genes, or an autotrophic gene which allows the host cell togrow in the absence of a nutrient for which the host cell strain isnormally autotrophic.

Representative examples of appropriate hosts include, but are notlimited to, bacterial cells, such as E. coli, Streptomyces andSalmonella typhimurium cells; fungal cells, such as yeast cells andinsect cells such as Drosophila S2 and Spodoptera Sf9 cells. Appropriateculture mediums and conditions for the above-described host cells areknown in the art.

If the vector is intended to be maintained in the host cellextrachromosomally, it will contain, in addition and origin ofreplication which will allow it to replicate in the host cell.Alternatively, if it is desired that the vector integrate into thechromosome, the vector is constructed such that it cannot replicate inthe host cell. For example, such a vector might be capable ofpropagation in another organism, for example, E. coli, but lack theproper origin of replication to be propagated in Corynebacterium. Inanother aspect of this embodiment, the vector is a shuttle vector whichcan replicate and be maintained in more than one host cell species, forexample, such a shuttle vector might be capable of replication in aCorynebacterium host cell such as a C. glutamicum host cell, and also inan E. coli host cell.

In one embodiment of the invention, the additional copies of theL-lysine biosynthesis pathway gene(s) selected from ask, asd, dapB, ddh,ORF2 and lysA can be integrated into the chromosome. Another embodimentof the invention provides that the additional copies of the L-lysinebiosynthesis pathway gene(s) are carried extra-chromosomally.Amplifications by a factor of 5 or less can be obtained by introducingthe additional gene copies into the chromosome of the host strain by wayof single event homologous recombination. In a most preferredembodiment, the recombination event results in the introduction of oneadditional copy of the copy of the gene or genes of interest. If morethan 5 copies of the genes are desired, multicopy plasmids carrying therecombinant DNA construct of the invention can be utilized.

In another embodiment of the invention, enzyme activity is increased byoverexpressing one or more genes of the group comprising ask, asd, dapB,ddh, ORF2 and lysA encoding one or more lysine biosynthetic pathwayenzymes. In one embodiment of the invention, said one or more genes areoperably linked directly or indirectly to one or more promotersequences. In another embodiment of the invention, said operably linkedpromoter sequences are heterologous, endogenous, or hybrid. In apreferred embodiment of the invention, said promoter sequences are oneor more of: a promoter sequence from the 5′ end of genes endogenous toC. glutamicum, a promoter sequence from plasmids that replicate in C.glutamicum, and, a promoter sequence from the genome of phage whichinfect C. glutamicum. In another embodiment, one or more of saidpromoter sequences are modified. In another preferred embodiment, saidmodification comprises truncation at the 5′ end, truncation at the 3′end, non-terminal insertion of one or more nucleotides, non-terminaldeletion of one or more nucleotides, addition of one or more nucleotidesat the 5′ end, addition of one or more nucleotides at the 3′ end, and,combinations thereof. In a preferred embodiment, the P1 promoter, thefirst promoter of the argS-lysA operon is used as the promoter for thelysA gene.

Alternative gene promoter elements can be utilized in the constructs ofthe invention. For example, known bacterial promoters suitable for thisuse in the present invention include the E. coli lacI and lacZpromoters, the T3 and T7 promoters, the gpt promoter, the lambda PR andPL promoters, the trp promoter, or promoters endogenous to the bacterialcells of the present invention. Other promoters useful in the inventioninclude regulated promoters, unregulated promoters and heterologouspromoters. Many such promoters are known to one of skill in the art. SeeSambrook, E. F. et al., Molecular Cloning: A Laboratory Manual, 2d ed.,Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989).

In addition, the vector can contain control regions that regulate aswell as engender expression. Generally, such regions will operate bycontrolling transcription, such as inducer or repressor binding sitesand enhancers, among others.

In a preferred embodiment of the invention, the KDB polynucleotidemolecule is encompassed in vector pDElia2_(FC5)-KDB. In anotherpreferred embodiment, the KDBH polynucleotide molecule is encompassed invector pK184-KDBH. In another preferred embodiment, the KDB2polynucleotide molecule is encompassed in vector pDElia2_(FC5)-KDB2. Ina further preferred embodiment, the KDB2HL polynucleotide is encompassedin vector pDElia2_(FC5)-KDB2HL.

It is a further object of the invention to provide a host cellcomprising a vector comprising any one of the isolated KDB, KDBH, KDB2or KDB2HL polynucleotide molecule.

Introduction of the construct into the host cell can be effected bycalcium phosphate transfection, DEAE-dextran mediated transfection,cationic lipid-mediated transfection, electroporation, transduction,infection, or other methods. Such methods are described in many standardlaboratory manuals, such as Davis et al., Basic Methods In MolecularBiology (1986). If the vector is a virus, it can be packaged in vitrousing an appropriate packaging cell line and then transduced into hostcells.

Representative examples of appropriate hosts for the above describedisolated nucleic acid molecules include, but are not limited to,bacterial cells, such as C. glutamicum, Escherichia coli, Streptomycesand Salmonella typhimurium cells; and fungal cells, such as yeast cells.Appropriate culture media and conditions for the above-described hostcells are known in the art.

Bacterial cells, such as E. coli and coryneform bacteria are preferredas host cells. Particularly preferred Corynebacterium and Brevibacteriumspecies of the methods and processes of the invention include:Corynebacterium glutamicum, Brevibacterium flavum, Brevibacteriumlactofermentum and other Cornynebacteria and Brevibacteria species knownin the art.

As will be understood by those skilled in the art, the term“Corynebacterium species” includes those organisms previously identifiedin the literature as “Brevibacterium species,” for exampleBrevibacterium flavum and Brevibacterium lactofermentum which have nowbeen reclassified into the genus Corynebacterium (Int. J. Syst.Bacteriol. 41: 255 (1981)).

It is a further object to provide a host cell wherein said host cell isa Brevibacterium selected from the group consisting of Brevibacteriumflavum NRRL-B30218, Brevibacterium flavum NRRL-B30458, Brevibacteriumflavum NRRL-B30410, Brevibacterium flavum NRRL-B30459, Brevibacteriumflavum NRRL-B30522, Brevibacterium flavum NRRL-B30219, Brevibacteriumlactofermentum NRRL-B30220, Brevibacterium lactofermentum NRRL-B30221,Brevibacterium lactofermentum NRRL-B30222, Brevibacterium flavumNRRL-30234 and Brevibacterium lactofermentum NRRL-30235. In anotherembodiment, the host cell is Escherichia coli. In a preferredembodiment, the host cell is E. coli DH5 α MCR NRRL-B30228. In anotherembodiment, the host cell is a C. glutamicum selected from the groupconsisting of C. glutamicum NRRL-B30236 and C. glutamicum NRRL-B30237.

The methods to increase the production of lysine and the processes forthe production of lysine of the invention can both utilize a steprequiring the transformation of an isolated nucleic acid molecule into ahost cell.

The methods to increase the production of lysine and the processes forthe production of lysine of the invention can utilize a step requiringamplification of at least one lysine biosynthesis pathway gene. As knownto one skilled in the art, the term amplification means increasing thenumber of a gene or genes of lysine biosynthetic pathway by any meansknown in the art. Particularly preferred means of amplification include:(1) the addition an isolated KDB, KDBH, KDB2 or KDB2HL polynucleic acidmolecule by insertion into the chromosome of a host cell, for example byhomologous recombination, and (2) the addition an isolated KDB, KDBH,KDB2 or KDB2HL polynucleic acid molecule into a host cell by way of aself-replicating, extra-chromosomal vector, for example, a plasmid.

Methods of inserting an isolated nucleic acid molecule into thechromosome of a host cell are known to those skilled in the art. Forexample, insertion of isolated nucleic acid molecules into thechromosome of Corynebacterium species can be done utilizing the pK184plasmid described by Jobling, M. et al., Nucleic Acids Research 18(17):5315-5316 (submitted 1990). Because these vectors lack a Corynebacteriumspecies origin of replication and contain a selectable marker such askanamycin (kan), cells will only be capable of growing under selectionif the vector has been inserted into the host cell chromosome byhomologous recombination.

In alternative embodiments, the invention also provides methods forincreasing lysine production and processes for the production of lysinewherein biosynthetic pathway gene amplification is accomplished throughthe introduction into a host cell of a self-replicating,extra-chromosomal vector, e.g., a plasmid, comprising an isolated KDB,KDBH, KDB2 or KDB2HL polynucleotide molecule. Suitable plasmids forthese embodiments include pSR1 and other derivatives of pSR1 (Archer, J.et al., J. Gen. Microbiol. 139: 1753-1759(1993)).

For various embodiments of the invention drawn to a method to increaseproduction of L-lysine, screening for increased production of L-lysine,can be determined by directly comparing the amount of L-lysine producedin culture by a Corynebacterium species host strain to that of aCorynebacterium species transformed host strain in which lysinebiosynthesis gene or genes are amplified. The level of production oflysine can conveniently be determined by the following formula tocalculate the percent yield from dextrose: [(g lysine/L/(g dextroseconsumed/L)]*100.

In one embodiment, the invention provides a method to increase theproduction of lysine comprising: (a) transforming a host cell with anisolated KDBH polynucleotide molecule (b) selecting a transformed hostcell; and (c) screening for increased production of lysine from saidtransformed host cell relative to said host cell. In another embodimentof the method, the method further comprises growing said transformedhost cell in a medium; and purifying lysine produced by said transformedhost cell.

A variety of media known to those skilled in the art can be used tosupport cell growth for the production of lysine. Illustrative examplesof suitable carbon sources include, but are not limited to:carbohydrates, such as glucose, fructose, sucrose, starch hydrolysate,cellulose hydrolysate and molasses; organic acids, such as acetic acid,propionic acid, formic acid, malic acid, citric acid, and fumaric acid;and alcohols, such as glycerol. Illustrative examples of suitablenitrogen sources include, but are not limited to: ammonia, includingammonia gas and aqueous ammonia; ammonium salts of inorganic or organicacids, such as ammonium chloride, ammonium phosphate, ammonium sulfateand ammonium acetate; and other nitrogen-containing sources, includingmeat extract, peptone, corn steep liquor, casein hydrolysate, soybeancake hydrolysate, urea and yeast extract.

A variety of fermentation techniques are known in the art which can beemployed in processes of the invention drawn to the production of aminoacids. Generally, amino acids can be commercially produced from theinvention in fermentation processes such as the batch type or of thefed-batch type. In batch type fermentations, all nutrients are added atthe beginning of the fermentation. In fed-batch or extended fed-batchtype fermentations one or a number of nutrients are continuouslysupplied to the culture, right from the beginning of the fermentation orafter the culture has reached a certain age, or when the nutrient(s)which are fed were exhausted from the culture fluid. A variant of theextended batch of fed-batch type fermentation is the repeated fed-batchor fill-and-draw fermentation, where part of the contents of thefermenter is removed at some time, for instance when the fermenter isfull, while feeding of a nutrient is continued. In this way afermentation can be extended for a longer time.

Another type of fermentation, the continuous fermentation or chemostatculture, uses continuous feeding of a complete medium, while culturefluid is continuously or semi-continuously withdrawn in such a way thatthe volume of the broth in the fermenter remains approximately constant.A continuous fermentation can in principle be maintained for an infinitetime.

In a batch fermentation an organism grows until one of the essentialnutrients in the medium becomes exhausted, or until fermentationconditions become unfavorable (e.g., the pH decreases to a valueinhibitory for microbial growth). In fed-batch fermentations measuresare normally taken to maintain favorable growth conditions, e.g., byusing pH control, and exhaustion of one or more essential nutrients isprevented by feeding these nutrient(s) to the culture. The microorganismwill continue to grow, at a growth rate dictated by the rate of nutrientfeed. Generally a single nutrient, very often the carbon source, willbecome limiting for growth. The same principle applies for a continuousfermentation, usually one nutrient in the medium feed is limiting, allother nutrients are in excess. The limiting nutrient will be present inthe culture fluid at a very low concentration, often unmeasurably low.Different types of nutrient limitation can be employed. Carbon sourcelimitation is most often used. Other examples are limitation by thenitrogen source, limitation by oxygen, limitation by a specific nutrientsuch as a vitamin or an amino acid (in case the microorganism isauxotrophic for such a compound), limitation by sulphur and limitationby phosphorous.

Lysine can be recovered by any method known in the art. Exemplaryprocedures are provided in the following: Van Walsem, H. J. & Thompson,M. C., J. Biotechnol. 59:127-132 (1997), and U.S. Pat. No. 3,565,951,both of which are incorporated herein by reference.

The pDElia2_(FC5)-KDB, the pK184-KDBH, the pDElia2_(FC5)-KDB2 and thepDElia2_(FC5)-KDB2HL constructs in NRRL-B11474 host cells were depositedat an acceptable International Depositary Authority in accordance withthe Budapest Treaty on the International Recognition of the Deposit ofMicroorganisms for the Purposes of Patent Procedure. The deposits havebeen made with the Agricultural Research Service, Culture Collection(NRRL), 1815 North University Street, Peoria, Ill. 61604.

All patents and publications referred to herein are expresslyincorporated by reference in their entirety.

EXAMPLES Example 1 Preparation of L-Lysine Pathway Multi-Gene Constructs

Constructs comprising a KDB, a KDBH, a KDB2HL or a KDB2 polynucleotidemolecule were made from the following sources: Source Gene(s) ask-asdStrain ATCC 21529; dapB Strain NRRL B11474; ddh Strain NRRL B11474; ORF2Strain NRRL B11474; lysA Strain ASO19; Promoter P1 argS-lysA operon frompRS6

The polymerase chain reaction (PCR) technique was used to construct theKDB, KDBH, KDB2, and KDBHL constructs. Standard PCR and subcloningprocedures were utilized in cloning the coding regions of ask-asd,dapB-ORF2-dapA, ddh.

The primers utilized for cloning experiments included: Primer nameSequence ask 5′-GGGTACCTCGCGAAGTAGCACCTGTCAC-3′ asd5′-GCGGATCCCCCATCGCCCCTCAAAGA-3′ dapB 5′-AACGGGCGGTGAAGGGCAACT-3′ ORF25′-GCTCATAGAGTTCAAGGTTACCTTCTTCCC-3′ ddh1 5′-CCATGGTACCAAGTGCGTGGCGAG-3′ddh2 5′-CCATGGTACCACACTGTTTCCTTGC-3′ lysA_((ATG))5′-CCGGAGAAGATGTAACAATGGCTAC-3′ lysA3B 5′-CCTCGACTGCAGACCCCTAGACACC-3′dapA 5′-TGAAAGACAGGGGTATCCAGA-3′

Construction procedures and intermediate plasmids are described in FIGS.11-14. The following steps (FIG. 11) were performed in constructing thepDElia2_(FC5)-KDB vector:

-   -   1. pGEMT-ask-asd: an approximately 2.6 Kb PCR product containing        the ask-asd operon of ATCC21529 using primers ask and asd was        cloned into pGEM-T (Promega pGEM-T vector systems).    -   2. pFC3-ask-asd: an approximately 2.6 Kb NsiI-ApaI fragment of        pGEMT-ask-asd was cloned into pFC3 cut with PstI and ApaI.    -   3. pFC3-dapB-ORF2-dapA: an approximately 2.9 Kb PCR product of        NRRL-B11474 dapB-ORF2-dapA coding region was cloned into pFC3 at        the EcoRV site.    -   4. pFC3-dapB: the large ClaI fragment of pFC3-dapB-ORF2-dapA was        religated.    -   5. pUC18-ddh: an approximately 1.3 Kb KpnI fragment of pADM21        containing ddh (NRRL-B11474 locus) was subcloned into pUC18 at        the KpnI site.    -   6. pFC1-ddh: an approximately 1.3 Kb SalI-EcoRI fragment of        pUC18-ddh was cloned into pFC1 cut with SalI and EcoRI.    -   7. pFC1-ddh-lysA: an approximately 2.1 Kb EcoRI-PstI fragment        (containing the intact lysA DNA) of pRS6 was cloned into        pFC1-ddh cut with EcoRI and PstI.    -   8. pFC1-ask-asd-ddh-lysA: an approximately 2.6 Kb SwaI-FseI        fragment of pFC3-ask-asd was cloned into pFC1-ddh-lysA cut with        SwaI and FseI.    -   9. pFC3-ask-asd-dapB-ddh-lysA: an approximately 6 Kb SpeI        fragment of pFC1-ask-asd-ddh-lysA was cloned into pFC3-dapB at        the SpeI site.    -   10. pDElia2_(FC5)-ask-asd-dapB-ddh-lysA (pDElia2_(FC5)-KDBHL):        an approximately 7.38 Kb NotI-PmeI fragment of        pFC3-ask-asd-dapB-ddh-lysA was cloned into pDElia² _(FC5) cut        with NotI and PmeI.    -   11. pDElia2: an approximately 1.24 Kb blunted PstI fragment of        pUC4K was ligated with the approximately 1.75 Kb DraI-SspI        fragment of pUC19.    -   12. pDElia2_(FC5): the small PvuII fragment of pFC5 was ligated        with the large PvuII fragment of pDElia2.    -   13. pDElia2_(FC5)-ask-asd-dapB (pDElia2_(FC5)-KDB): an        approximately 4 Kb ApaI fragment of pDElia2_(FC5)-KDBHL was        cloned into pDElia2_(FC5) at the ApaI site.

Corynebacterium (NRRL-B11474) containing the pDElia2_(FC5)-KDB constructwas deposited at an acceptable International Depositary Authority inaccordance with the Budapest Treaty on the International Recognition ofthe Deposit of Microorganisms for the Purposes of Patent Procedure. Thedeposit has been made with the Agricultural Research Service, CultureCollection (NRRL), 1815 North University Street, Peoria, Ill. 61604 onFeb. 1, 2001. The deposit is numbered NRRL-B30458.

The following steps (FIG. 12) were preformed in constructing thepK184-KDBH construct:

-   -   1. pGEMT-ask-asd: an approximately 2.6 Kb PCR product containing        the ask-asd operon of ATCC21529 using primers ask and asd was        cloned into pGEM-T (Promega pGEM-T vector systems).    -   2. pFC3-ask-asd: an approximately 2.6 Kb NsiI-ApaI fragment of        pGEMT-ask-asd was cloned into pFC3 cut with PstI and ApaI.    -   3. pFC3-ask-asd-ddh: an approximately 1.3 Kb KpnI fragment        containing NRRL-B11474 ddh was cloned into pFC3-ask-asd at the        KpnI site.    -   4. pFC3-dapB-ORF2-dapA: an approximately 2.9 Kb PCR product of        NRRL-B11474 dapB-ORF2-dapA coding region was cloned into pFC3 at        the EcoRV site.    -   5. pFC3-dapB: the large ClaI fragment of pFC3-dapB-ORF2-dapA was        religated.    -   6. pFC3-ask-asd-dapB-ddh: an approximately 4 Kb NotI-SwaI        fragment of pFC3-ask-asd-ddh was cloned into pFC3-dapB digested        with NotI and SmaI.    -   7. pK184-ask-asd-dapB-ddh (pK184-KDBH): an approximately 5.3 Kb        PmeI fragment containing ask-asd-dapB-ddh was cloned into pK184        at the SmaI site.

Corynebacterium (NRRL-B11474) containing the pK184-KDBH construct wasdeposited at an acceptable International Depositary Authority inaccordance with the Budapest Treaty on the International Recognition ofthe Deposit of Microorganisms for the Purposes of Patent Procedure. Thedeposit has been made with the Agricultural Research Service, CultureCollection (NRRL), 1815 North University Street, Peoria, Ill. 61604 onFeb. 1, 2001. The deposit is numbered NRRL-B30410.

The following steps (FIG. 13) were performed in constructing thepDElia2_(FC5)-KDB2 vector:

-   -   1. pGEMT-ask-asd: an approximately 2.6 Kb PCR product containing        the ask-asd operon of ATCC21529 using primers ask and asd was        cloned into pGEM-T (Promega pGEM-T vector systems).    -   2. pUC18-ddh: an approximately 1.3 Kb KpnI fragment of pADM21        containing ddh (BF100 locus) was subcloned into pUC18 at the        KpnI site.    -   3. pFC3-ask-asd: an approximately 2.6 Kb NsiI-ApaI fragment of        pGEMT-ask-asd was cloned into pFC3 cut with PstI and ApaI    -   4. pFC1-dapB-ORF2: an approximately 2 Kb PCR product of        NRRL-B11474 dapB-ORF2 coding region was cloned into pFC1 at the        EcoRV site.    -   5. pFC1-ddh: an approximately 1.3 Kb PstI-EcoRI fragment of        pUC18-ddh was cloned into pFC1 cut with PstI and EcoRI.    -   6. pUC19-P1: an approximately 550 bp HpaI-PvuII fragment        (containing the first promoter, P1, of the argS-lysA operon) of        pRS6 was cloned into pUC19 at the SmaI site.    -   7. pUC19-P1lysA: an approximately 1.45 Kb promoterless PCR        product, using primers LysA(ATG) and LysA3B, of ASO19 lysA        coding region is cloned into pUC19-P1 at the HincII site.    -   8. pFC1-P1lysA: an approximately 2 Kb EcoRI-HindIII fragment of        pUC19-P1lysA was cloned in to pFC1 cut with EcoRI and HindIII.    -   9. pFC1-ddh-P1lysA: an approximately 1.3 Kb EcoRI-NotI fragment        of pFC1-ddh was cloned into pFC1-P1lysA cut with EcoRI and NotI.    -   10. pFC1-ask-asd-ddh-P1lysA: an approximately 2.6 Kb SwaI-FseI        fragment of pFC3-asd-asd was cloned into pFC1-ddh-P1lysA cut        with SwaI and FseI.    -   10. pFC1-ask-asd-dapB-ORF2-ddh-P1lysA (pFC1-KDB2HPIL): an        approximately 5.9 Kb SpeI fragment of pFC1-ask-asd-ddh-P1lysA        was cloned into pFC1-dapB-ORF2 at the SpeI site.    -   11. pDElia2_(FC5): the small PvuII fragment of pFC5 was ligated        with the large PvuII fragment of pDElia2.    -   12. pDElia2_(FC5)-ask-asd-dapB-ORF2 (pDElia2_(FC5)-KDB2): an        approximately 4.7 Kb ApaI fragment containing KDB2 of        pFC1-KDB2HP1L was cloned into pDElia2_(FC5) at the ApaI site.

Corynebacterium (NRRL-B11474) containing the pDElia2_(FC5)-KDB2construct was deposited at an acceptable International DepositaryAuthority in accordance with the Budapest Treaty on the InternationalRecognition of the Deposit of Microorganisms for the Purposes of PatentProcedure. The deposit has been made with the Agricultural ResearchService, Culture Collection (NRRL), 1815 North University Street,Peoria, Ill. 61604 on Feb. 1, 2001. The deposit is numbered NRRL-B30459.

The following steps (FIG. 14) were performed in constructing thepDElia2_(FC5)-KDB2HP1L vector:

-   -   1. pGEMT-ask-asd: an approximately 2.6 Kb PCR product containing        the ask-asd operon of ATCC21529 using primers ask and asd was        cloned into pGEM-T (Promega pGEM-T vector systems).    -   2. pUC18-ddh: an approximately 1.3 Kb KpnI fragment of pADM21        containing ddh (NRRL-B11474 locus) was subcloned into pUC18 at        the KpnI site.    -   3. pFC3-ask-asd: an approximately 2.6 Kb NsiI-ApaI fragment of        pGEMT-ask-asd was cloned into pFC3 cut with Pst1 and ApaI.    -   4. pFC1-dapB-ORF2: an approximately 2 Kb PCR product of        NRRL-B11474 dapB-ORF2 coding region was cloned into pFC1 at the        EcoRV site.    -   5. pFC1-ddh: an approximately 1.3 Kb PstI-EcoRI fragment of        pUC19-ddh was cloned into pFC1 cut with PstI and EcoRI.    -   6. pUC19-P1: an approximately 550 bp HpaI-PvuII fragment        (containing the first promoter, P1, of the argS-lysA operon) of        pRS6 was cloned into pUC19 at the SmaI site.    -   7. pUC19-P1lysA: an approximately 1.45 Kb promoterless PCR        product, using primers LysA(ATG) and LysA3B, of ASO19 lysA        coding region is cloned into pUC19-P1 at the HincII site.    -   8. pFC1-P1lysA: an approximately 2 Kb EcoRI-HindIII fragment of        pUC19-P1lysA was cloned into pFC1 cut with EcoRI and HindIII.    -   9. pFC1-ddh-P1lysA: an approximately 1.3 Kb EcoRI-NotI fragment        of pFC1-ddh was cloned into pFC1-P1lysA cut with EcoRI and NotI.    -   10. pFC1-ask-asd-ddh-P1lysA: an approximately 2.6 Kb SwaI-FseI        fragment of pFC3-ask-asd was cloned into pFC1-ddh-P1lysA cut        with SwaI and FseI.    -   11. pFC1-ask-asd-dapB-ORF2-ddh-P1lysA (pFC1-KDB2HPIL): an        approximately 5.9 Kb SpeI fragment of pFC1-ask-asd-ddh-P1lysA        was cloned into pFC1-dapB-ORF2 at the SpeI site.    -   12. pDElia2_(FC5): the small PvuII fragment of pFC5 was ligated        with the large PvuII fragment of pDElia2    -   13. pDElia2_(FC5)-ask-asd-dapB-ORF2-ddh-P1lysA        (pDElia2_(FC5)-KDB2HP1L): an approximately 7.9 Kb NHE fragment        of pFC1-ask-asd-dapB-ORF2-ddh-P1lysA was cloned into        pDElia2_(FC5) at the NHE site.

Corynebacterium (NRRL-B11474) containing the pDElia2_(FC5)-KDB2HP1Lconstruct was deposited at an acceptable International DepositaryAuthority in accordance with the Budapest Treaty on the InternationalRecognition of the Deposit of Microorganisms for the Purposes of PatentProcedure. The deposit has been made with the Agricultural ResearchService, Culture Collection (NRRL), 1815 North University Street,Peoria, Ill. 61604 on Feb. 1, 2001. The deposit is numbered NRRL-B30522.

Example 2 Screening and Selection of Strains with Improved L-LysineProduction

The production of L-lysine by cells stably transformed with multi-geneconstructs is summarized in Table 1. TABLE 1 Lysine production byvarious parental and stably transfected bacteria lysine titer L-lysineStrain Tested (g/L) yield (%) Cell Deposit NRRL-B11474 31 30NRRL-B11474::pDElia2_(FC5)-KDB 34 37 NRRL-B30458 NRRL-B11474 31 31NRRL-B11474::pK184-KDBH 38 37.4 NRRL-B30410 NRRL-B11474 30 30NRRL-B11474::pDElia2_(FC5)-KDB2 39 37 NRRL-B30459 NRRL-B11474 31 33NRRL-B11474::pDElia2_(FC5)-KDB2HP1L 38 41 NRRL-B30522

Having now fully described the present invention in some detail by wayof illustration and example for purposes of clarity of understanding, itwill be obvious to one of ordinary skill in the art that same can beperformed by modifying or changing the invention with a wide andequivalent range of conditions, formulations and other parametersthereof, and that such modifications or changes are intended to beencompassed within the scope of the appended claims.

All publications, patents and patent applications mentioned in thisspecification are indicative of the level of skill of those skilled inthe art to which this invention pertains, and are herein incorporated byreference to the same extent as if each individual publication, patentor patent application was specifically and individually indicated to beincorporated by reference.

1. An isolated polynucleotide molecule comprising: (a) a nucleic acidmolecule encoding an aspartate kinase (ask) polypeptide; (b) a nucleicacid molecule encoding an aspartate-semialdehyde dehydrogenase (asd)polypeptide; and (c) a nucleic acid molecule encoding adihydrodipicolinate reductase (dapB) polypeptide.
 2. The polynucleotidemolecule of claim 1, wherein said polynucleotide molecule additionallycomprises a nucleic acid encoding a complete or truncateddiaminopimelate dehydrogenase (ddh) polypeptide.
 3. The polynucleotidemolecule of claim 1, wherein said polynucleotide molecule additionallycomprises a nucleic acid encoding a complete or truncated ORF2polypeptide.
 4. The polynucleotide molecule of claim 1, wherein saidpolynucleotide molecule additionally comprises a nucleic acid encodingcomplete or truncated ddh, ORF2 and diaminopimelate decarboxylase (lysA)polypeptides.
 5. The polynucleotide molecule of claim 4, wherein saidpolynucleotide molecule additionally comprises a P1 promoter element ofSEQ ID NO:15.
 6. The polynucleotide molecule of claim 5, wherein said P1promoter element is adjacent to said nucleic acid encoding lysA.
 7. Thepolynucleotide molecule of claim 1, wherein said ask, asd and dapBpolypeptides are encoded by Corynebacterium, Brevibacterium flavum, orBrevibacterium lactovermentum.
 8. The polynucleotide molecule of claim1, wherein said ask and asd polypeptides are encoded by the ask-asdoperon of ATCC21529.
 9. The polynucleotide molecule of claim 2, whereinsaid ddh polypeptide is encoded by Corynebacterium, Brevibacteriumflavum or Brevibacterium lactovermentum.
 10. The polynucleotide moleculeof claim 3, wherein said ORF2 polypeptide is encoded by Corynebacterium,Brevibacterium flavum or Brevibacterium lactovermentum.
 11. Thepolynucleotide molecule of claim 4, wherein said lysA polypeptide isencoded by Corynebacterium, Brevibacterium flavum or Brevibacteriumlactovermentum.
 12. The polynucleotide molecule of claim 1, wherein saiddapB polypeptide is encoded by the coding region of the dapB gene ofNRRL-B11474.
 13. The polynucleotide molecule of claim 2, wherein saidddh polypeptide is encoded by the coding region of the ddh gene ofNRRL-B11474.
 14. The polynucleotide molecule of claim 3, wherein saidORF2 polypeptide is encoded by the coding region of the ORF2 gene ofNRRL-B11474.
 15. The polynucleotide molecule of claim 4, wherein saidlysA polypeptide is encoded by the coding region of the lysA gene ofASO19.
 16. A vector comprising the isolated polynucleotide molecule ofclaim
 1. 17. A host cell comprising said vector of claim
 16. 18. Thehost cell of claim 17, wherein said cell is a prokaryotic cell.
 19. Thehost cell of claim 17, wherein the cell is a eukaryotic cell.
 20. Thehost cell of claim 17, wherein said host cell is a Brevibacteriumflavum, Brevibacterium lactofermentum or Corynebacterium glutamicumcell.
 21. The host cell of claim 17 wherein said host cell is anEscherichia coli cell.
 22. A method for transforming a host cellcomprising: (a) transforming a host cell with the polynucleotidemolecule of claim 1, wherein said isolated polynucleotide molecule isstably integrated into said host cell's chromosome; and (b) selecting atransformed host cell.
 23. A method for transforming a host cellcomprising: (a) transforming a host cell with the polynucleotidemolecule of claim 1, wherein said isolated polynucleotide molecule ismaintained in said host cell as extrachromosomal DNA; and (b) selectinga transformed host cell.
 24. A method of producing lysine comprisingculturing said host cells of claim 17 in a culture medium, wherein saidhost cells produce lysine into said culture medium.
 25. Thepolynucleotide molecule of claim 1, wherein said polynucleotide moleculedoes not comprise a nucleic acid molecule encoding any one ofdihydrodipicolinate synthase (dapA), tetrahydrodipicolinate succinylase(dapD), N-succinylaminoketopimelate transaminase (dapC),N-succinyl-diaminopimelate desuccinylase (dapE) or diaminopimelateepimerase (dapF) polypeptides.